While the variable regions within the Fab (fragment antigen binding) domains of antibodies are responsible for the recognition of the antigen, the Fc (fragment crystallizable) region represents an invariant part of the antibody that is responsible for the mediation of effector functions. In the case of immunoglobulin G (IgG) these encompass the fixation of complement and the binding to Fcγ receptors (FcγRs). The presence of an N-linked oligosaccharide at a single conserved site (Asn297) within the CH2 domain of the homodimeric Fc fragment is mandatory for the mediation of both of these effector functions. It was only recently discovered that modification of the attached carbohydrates can also have an affinity improving effect for the interaction between FcγRIIIa and IgG. The carbohydrate modification responsible for this effect is the absence of a fucose residue which is usually attached to the first N-acetylglucosamine (GlcNAc) residue in the biantennary complex-type IgG glycan (FIG. 1).
It could be demonstrated by in vivo and in vitro experiments that such increased affinity results in enhanced antibody-dependent cellular cytotoxicity (ADCC) mainly mediated by natural killer (NK) cells. Consequently, it is also believed that such a-fucosylated antibodies have an improved efficacy in treatments that aim to eradicate opsonized cells.
The generation of a-fucosylated antibodies represents an important biotechnological challenge which can be achieved by several methods. While cell lines with a complete depletion of enzymes involved in the biosynthesis of fucosylation (e.g. by gene knockout) may yield quantitatively a-fucosylated antibodies, most other methods do not. For example, siRNA treatment or co-cultivation of antibody-expressing cells with kifunensine (Zhou et al., Biotechnol Bioeng (2008) 99, 652-665), as well as carbohydrate modification by N-acetylglucosaminyltransferase III (GnT-III), which promotes the formation of bisected oligosaccharides consequently inhibiting the fucosylation reaction (Umana et al., Nat Biotech (1999) 17, 176-180), lead to only partially a-fucosylated antibodies.
These partially a-fucosylated antibodies can principally exhibit a heterogeneous a-fucosylation distribution within a pool of antibodies. For example, fucosylation rates can be different during fermentation. Also, the event of fucosylation could be cooperative, i.e. the second fucosylation on the homodimeric antibody may occur with an increased (positive cooperativity) or decreased (negative cooperativity) rate compared to the first one.
The FcγRIIIa/IgG complex has a 1:1 stoichiometry but IgG has two binding sites for FcγRIIIa. Consequently, in a single a-fucosylated antibody the receptor can bind with high affinity to the binding site formed by the IgG's a-fucosylated glycan and protein core or with low affinity to the binding site consisting of the fucosylated carbohydrate and the protein core. It can therefore be concluded that a pool of antibodies with 50% a-fucosylation may consist of a homogeneous population of antibodies in which only one of the two N-glycans is fucosylated, or 50% of antibodies in which both N-glycans are fucosylated while in the other 50% none of the N-glycans are fucosylated. It is obvious that such a differential partition of a-fucosylation influences the overall affinity to FcγRIIIa and results in a different biological activity. It is therefore mandatory to analyze the biological activity of such an antibody preparation either directly by employing a biological test system (bioassay) or indirectly by biochemically measuring the rate and distribution of the a-fucosylation, which yields a more exact result.
The current state-of-the-art glycoanalytics uses N-glycosidase F (PNGase F) from Flavobacterium meningosepticum to cleave off the N-linked carbohydrates with a subsequent MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analysis (according to Papac et al., Glycobiology (1998) 8, 445-454). By employing such a process, however, the linkage information is lost and the determination of fucosylation distribution within an antibody preparation is not possible.
On the other hand, analysis of the complete antibody using ESI-MS (electrospray ionization mass spectrometry) yields complex mass patterns that do not allow a quantitative interpretation due to the various modifications other than fucosylation—like galactosylation, C-terminal lysine heterogeneity, deamidation etc.—that may or may not occur in both subunits of the homodimeric IgG.
Therefore, there is a need for a new analytical method that eliminates the mentioned heterogeneity but maintains the linkage information.